In the earlier research efforts conducted by one of the present inventors, a group of recombinant bacterial plasmid cloning vehicles for expression of exogenous genes in transformed bacterial hosts was constructed, comprising a DNA insert fragment coding for the desired polypeptide, linked in reading phase with one or more functional fragments derived from any outer membrane protein gene of any gram-negative bacterium. In a preferred embodiment of these expression plasmids, the exogenous DNA codes for mammalian hormones, enzymes or immunogenic proteins (or intermediates therefor), the functional fragments are derived from the lipoprotein gene of E. coli, and the desired polypeptide is expressed in E. coli transformants. In a more preferred embodiment, the DNA sequence coding for the desired protein is linked with and is expressed under the control of four specific functional fragments associated with the E. coli lipoprotein gene, namely, the promoter, the 5'-untranslated region, the 3'-untranslated region and the transcription termination site of that gene.
These expression plasmids may also include a second promoter, preferably an inducible promoter and most preferably a DNA sequence consisting of 95 base pairs ("bp") and containing the E. coli .beta.-galactosidase or "lac" promoter-operator, which is inserted immediately downstream of the lipoprotein promoter. This region serves not only as another transcription initiation site, but also as a repressor-binding site, thus working as a transcriptional "switch" for transcription initiated from the lipoprotein or "lpp" promoter, so that the exogenous DNA is expressed only in the presence of a "lactose inducer." When induced, the DNA coding for the desired polypeptide is transcribed from both promoters, thereby increasing the yield of the desired product over that obtained when expression is directed by the inducible lac promoter-operator alone. Accordingly, either constitutive or inducible gene expression may be achieved using these lpp gene cloning vehicles, provided that when inducible expression is desired, special E. coli strains are used as transformants, specifically, those which carry a mutant gene which overproduces the lactose repressor molecule.
The earlier research efforts of one of the present inventors also provided a modification of the foregoing scheme, namely, a class of "auto-regulated" inducible expression cloning vehicles, each member of the class being otherwise identical with its analog in the class of inducible expression vehicles described above, but further including the DNA sequence coding for the repressor molecule capable of binding with the inducible promoter used in the vehicle. In the preferred embodiment, the autoregulated expression plasmids incorporate an intact, functional E. coli lacI gene for this purpose. Since the expression of the exogenous genetic information in these cloning vehicles is regulated from within each cloning vehicle itself, a more effective transcriptional "switch" is provided, insuring that transcription is completely repressed in the absence of the appropriate inducer, without the necessity of utilizing special E. coli strains (which overproduce the repressor molecule) as transformants.
The earlier research of one of the present inventors revealed that the lipoprotein of E. coli is a "secretory" protein, i.e., it is produced from a precursor, which is then secreted across the cytoplasmic membrane and processed to the lipoprotein. In nature, translation of the lipoprotein mRNA transcript actually yields this precursor, called the prolipoprotein, which has a "peptide extension" or "signal peptide" at its amino terminus consisting of 20 amino acid residues, followed by the known 58 amino acid sequence of the lipoprotein. While the mechanisms involved in the secretion process are not yet well understood, the signal peptide is considered to direct the translocation in vivo of the prolipoprotein across the cytoplasmic membrane, in the process of which the peptide extension itself is removed, yielding mature lipoprotein.
Therefore, in light of the secretory nature of the lpp gene, each class of prior art expression plasmids (constitutive, inducible and "auto-regulated" inducible) most preferably includes three sub-classes of plasmids, the members of each subclass containing one of three alternative insertion sites for the exogenous genetic material. In this manner, the selection of a particular plasmid or a particular sub-class of plasmids for gene expression can influence the ultimate location at which the expression product can be found and collected. Using one of these insertion sites, for example, the desired polypeptide can be expressed with a leader sequence located at the amino terminal, the leader comprising the signal peptide of the E. coli lipoprotein, such that the desired product may be secreted through the cytoplasmic membrane and the signal peptide removed in vivo by processes native to the transformant, to yield the exogenous gene product. On the other hand, using expression plasmids which contain one or the other of the two remaining insertion sites, the expression product can be expected to be found either in the cytoplasm of the cell, or in the cell wall, respectively.
While the plasmids of each sub-class share a common insertion site, they differ from one another in their individual reading frames. Thus, each prior art sub-class comprises three plasmids, whose reading frames in effect differ by one base pair, enabling the selection of any desired reading frame for each insertion site and thereby facilitating the use of these expression vehicles with a wide variety of DNA insert fragments without the necessity of any direct modification of the reading frames of those fragments.
A further modification of the foregoing scheme was previously provided by the present inventors, in which the yield of the desired expression product was enhanced still further, without affecting the inducibility of the system, by varying slightly the size of the DNA fragment carrying the lac promoter-operator region. Specifically, by substituting a different, slightly longer DNA fragment, consisting of 105 bp but containing substantially the same, natural E. coli lac promoter-operator DNA sequence as contained in the 95 bp fragment described above, the amount of the desired polypeptide produced was found to increase dramatically.
Nevertheless, all of the prior art expression plasmids in which secretion of the desired polypeptide across the cytoplasmic membrane is achieved by fusing the E. coli lipoprotein signal peptide directly to the amino terminal end of the desired gene product, share a common disadvantage. The construction of these plasmids requires deletion of the DNA coding for the amino-terminal cysteine residue of the mature lipoprotein. While this structure permits synthesis of the desired polypeptide to commence immediately following the signal peptide, without any extraneous intervening amino acid residues, it also prevents the occurrence of "lipid modification," a natural transformation normally undergone by the native E. coli lipoprotein precursor (after synthesis but prior to secretion), for which the amino terminal cysteine residue is essential. This modification at the cleavage site is normally required to enable subsequent cleavage of the lipoprotein signal peptide.
As a consequence of the cysteine residue deletion, although gene products expressed with the prior art cloning vehicles (also called "vectors") are satisfactorily translocated across the cytoplasmic membrane (directed by the lipoprotein signal peptide), subsequent cleavage of that signal peptide by the cell's own lipoprotein-specific cleaving enzyme (called a "signal peptidase") does not occur because the alteration of the cleavage site normally introduced by the lipid modification is absent. Nevertheless, despite this limitation, cleavage of the lipoprotein signal peptide does occur when certain proteins are expressed, due to the presence of a second, non-lipoproteinspecific signal peptidase in E. coli cells, although this occurs only if the particular protein being expressed has the appropriate conformation, a physical attribute which is unknown and which cannot be predicted for most proteins.
Accordingly, the prior art lipoprotein secretion plasmids have limited application, since for production of most proteins, an extra, somewhat costly synthetic step is required to cleave the lipoprotein signal peptide from the remainder of the secreted moiety in order to obtain a pure form of the desired gene product. It is therefore the principal object of the present invention to provide a new class of plasmid cloning vehicles with which these disadvantages may be overcome.